ICT immunoassay for Legionella pneumophila serogroup 1 antigen employing affinity purified antibodies thereto

ABSTRACT

The present invention involves extracting from  Legionella  bacteria, particularly  L. pneumophila  bacteria, an essentially protein-free O-polysaccharide or carbohydrate antigen, coupling this antigen to an activated chromatographic column through a protein spacer molecule which is first conjugated to the antigen, utilizing the column thus prepared for the affinity purification of raw polyvalent antibodies to the same  Legionella  bacterium from which the O-polysaccharide or carbohydrate antigen was separated—thereby obtaining antigen-specific antibodies which are useful for the rapid detection of the corresponding  Legionella  bacterium or its antibody in human bodily fluids such as urine, sputum, blood and the like or in environmental samples suspected of harboring the  Legionella  bacterium.

This invention relates to essentially protein-free carbohydrate,including polysaccharide, antigens separated from bacteria of the genusLegionella, and especially from serogroups and/or strains of Legionellapneumophila, including the O-polysaccharide antigen of L. pneumophilaserogroup 1, and to the use of these antigens in the affinitypurification of polyvalent antibodies to corresponding Legionellaorganisms. More particularly, the invention encompasses coupling thecarbohydrate or polysaccharide antigen separated from a Legionellabacterium to an activated chromatographic column and using that columnfor affinity purification of the polyclonal antibodies to the samespecies, or the same serogroup of a species, of Legionella. Theinvention further encompasses the use of the affinity-purifiedpolyvalent antibodies produced in immunochemical assays for thedetection of Legionella-caused diseases such as Legionnaires disease andPontiac fever in human patients and for the detection of environmentalsources of Legionella infectious agents.

BACKGROUND

Legionnaires' disease, a pneumonia-like human infection caused bygram-negative bacteria of the genus Legionella, is virtually impossibleto differentiate on a reliable clinical basis (involving assessment ofpatient symptoms without laboratory tests) from pneumonia and othersimilar lung infections. Because the disease may produce lung abscesses,infections in other bodily organs or bacteremia, and its mortality rateis significantly increased by delay in commencing appropriate therapy,there is a need for rapid and reliable diagnostic tests which has todate been only partially met. Stout, J. E. and Yu, V. L.,“Legionellosis”, 337, N. Eng. J. Med. 682-687 (1997). Efforts to developsuch tests have been hampered by the fact that there are a number ofLegionella species, at least some of which are known to have a number ofdistinct serogroups.

Legionnaires' disease was first recognized in the summer of 1976 and anumber of techniques for detecting Legionella (“L.”) pneumophila, whichis now known to account for some 90% of cases (Stout and Yu, supra),have been developed in the interim. In general, these tests aretime-consuming and incapable of identifying more than one serogroup ofthe 15 serogroups known so far to fall within the L. pneumophilaspecies. It should be noted, however, that, as reported by Stout and Yu,supra, more than 80% of reported cases of Legionnaires' disease areattributable to L. pneumophila serogroup 1—a fact which makes thedevelopment of a rapid, reliable immunoassay for that entity ofparticular importance and has led researchers to focus on this as apriority.

The early efforts to establish the identity of the causative agent ofLegionnaires' disease depended largely upon culturing the bacteria for 5to 6 days and examining the culture microscopically. Efforts to speed upthe identification process led to numerous immunochemical tests ofvarying sensitivity and specificity including, inter alia, the presentlycommercially available EQUATE™ radioimmunoassay (“RIA”), and the Binaxenzyme immunoassay (“EIA”), both of which are sold in kit form byapplicants' assignee, Binax, Inc. As indicated by Hackman, B. A. et al.in “Comparison of Binax Legionella Urinary Antigen EIA Kit with BinaxRIA Urinary Antigen Kit for Detection of Legionella pneumophilaSerogroup I Antigen”, 34 J. Clin. Microbiol. 1579-1580 (1996), both ofthese assays were found to be specific for L. pneumophila Serogroup Iantigen. The reported sensitivity of the EIA was 77%; that of the RIAwas higher. The article indicates that each can be performed within“less than 3 h[ours] from beginning to end”; in Binax's own tests eachrequires at least 2½ hours to perform. More information about this EIAassay appears in Kazandjian, D. et al., “Rapid Diagnosis of Legionellapneumophila Serogroup I Infection with Binax Enzyme Immunoassay UrinaryAntigen Test”, 35 J. Clin. Immunobiol. 954-956 (1997).

BRIEF DESCRIPTION OF THE INVENTION

Perhaps the most significant advantage of the immunochromatographic test(“ICT”) described herein is its ability to give a test result within a15-minute time span for the presence or absence of L. pneumophilaSerogroup I (or its antigen), which result is of high specificity andsensitivity. The speed with which this test can be reliably conducted toyield a result of high specificity and sensitivity is believed to be dueto the strongly reactive nature of the affinity purified antibodiesprepared in accordance with this invention. The superior reactiveproperties of these antibodies, in turn, is believed to be attributableto the use for affinity purification of the novel, essentiallyprotein-free O-polysaccharide antigen of L. pneumophila serogroup 1which is also a part of this invention.

Another ICT test has been made possible by the separation, according tothis invention, of an antigen of L. pneumophila serogroup 5 which is ofnon-proteinaceous, carbohydrate nature and is common to multipleserogroups of L. pneumophila. The presence of such a common antigen hasbeen suggested in the scientific literature; see e.g., Nolte, F. S. etal., “Electrophoretic and Serological Characterization of theLipopolysaccharides of Legionella pneumophila”, 52 Infection andImmunity 676-681 (1986); Otten, S. et al., “Serospecific Antigens ofLegionella pneumophila”, 167 J. Bacteriol. 893-904 (1986); Barthe, C. etal., “Common Epitope on the Lipopolysaccharide of Legionella pneumophilaRecognized by a Monoclonal Antibody”, 26 J. Clin. Microbiol. 1016-1023(1988); Knirel, Y. A. et al., “The Structure of the O-specific Chain ofLegionella pneumophila Serogroup I”, 227 Eur. J. Biochem. 239-245(1994). Heretofore there has been no clear report of any separation ofsuch an antigen for use in developing a useful broad spectrumimmuno-chemical assay.

This invention comprises the extraction from any Legionella bacterium,and especially from a bacterium of any of the serogroups of L.pneumophila of an essentially protein-free carbohydrate antigen, thepreparation of a conjugate of this antigen with a spacer protein, thecoupling of the conjugate to an affinity column, the use of that columnfor the purification of polyvalent antibodies to the correspondingLegionella bacterium, such as a bacterium of a serogroup of L.pneumophila and the use of the antibodies thus purified in an ICTimmunoassay for detecting Legionella bacteria or their antigens,including antigens of L. pneumophila serogroups or their correspondingbacteria, as more specifically described hereinafter.

In particular, the invention includes the separation of an essentiallyprotein-free O-polysaccharide antigen specific to L. pneumophilaserogroup 1, its use in the affinity purification of the polyvalentantibody specific to the same microorganism and the use of that affinitypurified antibody in an ICT immunoassay of high specificity and highsensitivity that is performable within 15 minutes.

In another embodiment, the invention includes the separation from L.pneumophila serogroup 5 of an essentially protein-free carbohydrateantigen, and its use in the affinity purification of the polyclonalantibody specific to the L. pneumophila serogroup 5 antigen (i.e., anantibody that was obtained from a rabbit immunized with the saidantigen); this antibody showed an ability to cross-react with antigensof the L. pneumophila serotypes 1, 2 and 4 in addition to the antigen ofserotype 5.

DESCRIPTION OF THE DRAWINGS

FIG. 1 and its related FIGS. 1A, 1B and 1C hereof show the structure ofa typical ICT device which is suitably adapted to perform the L.pneumophila serogroup 1 specific assay, as described in Examples VII,VIII and IX hereof.

FIG. 2 hereof shows the results of Western blot analyses ofphosphate-buffered saline extracts of L. pneumophila serogroups 1, 2, 4and 5 with L. pneumophila serogroup 5, essentially protein-freecarbohydrate antigen-affinity-purified polyclonal antibody specific toserogroup 5 antigen, which analysis is described in Example X hereof.

DETAILED DESCRIPTION OF THE INVENTION

Previous experience in the art, including experience with the Binax RIAassay for L. pneumophila Serogroup 1 antigen sold under the trademarkEQUATE and the Binax EIA assay for the same antigen, has shown thatantigens of the Legionella species, including antigens of various L.pneumophila serogroups, are more conveniently detectable in specimensfrom the urine of patients infected with a Legionella microorganism thanin specimens of blood, sputum or other fluids. This is in part becausethe Legionella antigens usually appear in urine within 1-3 days afterinfection of a human patient whereas their appearance at a detectablelevel in blood may occur later, and also in part because patientsinfected with Legionnaires' disease often do not produce much sputum.The ICT test which forms a part of this invention can be configured torun on blood, sputum or some other fluid such as cerebrospinal fluid, oron aqueous samples of environmental origin. It is noted that urine isgenerally the preferred sample fluid for diagnosis of human patientsbecause it can be obtained non-invasively and easily, even in thedoctor's office, and it si not as readily contaminated with othermicroorganisms, e.g., oral microflora present in sputum, which areinnocuous but may affect the results obtained.

-   -   Broadly speaking, the ICT test for L. pneumophila serogroup 1        antigen that is specifically a part of the present invention may        be designed to be run in any known disposable ICT device        disclosed in the art. Preferably, the test is conducted using an        ICT device of the type disclosed in copending U.S. application        Ser. No. 07/706,639 now U.S. Pat. No. 6,168,956 of Howard        Chandler, or one of its continuation-in part applications, all        assigned to Smith-Kline Diagnostics, INC. but exclusively        licensed to Binax, Inc. in a wide area of applications that        includes the present diagnostic field—i.e., diseases of the        respiratory system. The device is suitably impregnated with the        affinity purified polyvalent antibodies herein disclosed which        are specific to the antigen of L. pneumophila serogroup 1        antigen. Positive results of the assay are shown by the        appearance of a color upon reaction of suitably labeled        antibodies with the antigen. Suitable labels may be any those        known in the art to produce visible color when antibodies        conjugated thereto react with antigen, including finely divided        metallics and various labeling material. Colloidal gold is the        preferred label.

The invention contemplates that essentially protein free carbohydrateantigens may similarly be separated from each of the Legionellabacteria, including from bacteria of other Legionella species and frombacteria of other serogroups of L. pneumophila, that such antigens maysimilarly be utilized in the affinity purification of polyvalentantibodies to the corresponding Legionella species or serogroup bacteriaand its antigen, and that these affinity-purified antibodies may beutilized in ICT and other immunoassays as specifically disclosed hereinfor affinity-purified antibodies to L. pneumophila serogroup 1.

Preliminary to the preparation of the device is obtaining the antibodiesand effecting their affinity purification. The antibodies that may beused in this invention are conventional polyvalent (also called“polyclonal”) antibodies obtained by the well known process ofimmunizing a rabbit, goat or other animal to a Legionella antigen, e.g.,L. pneumophila antigen of known serogroup and bleeding the animal afterthe passage of an appropriate time period to obtain serum containing thedesired antibodies. See, e.g., Cherry, U. B. and McKinney, R. M., pp.91-104 in Jones, G. L. and Herbert, G. A. (Eds.), “Legionnaires” thedisease, the bacterium and the methodology, (Center for Disease Control,Atlanta, 1979). In this invention, relative to the ICT immunoassay forL. pneumophila serogroup 1 disclosed herein, the rabbit or other animalis immunized to L. pneumophila serogroup 1 antigen and the polyvalentantibodies recovered from its blood are antibodies to L. pneumophilaserogroup 1.

The polyvalent antibodies to be used in this invention are furthersubjected to affinity purification on a specially preparedchromatographic column which employs the essentially protein-freecarbohydrate, including polysaccharide, antigen of the same bacteriumagainst which the antibodies are reactive as the purifying agent forthem.

Preliminary to the affinity purification of the antibodies, it istherefore necessary according to this invention to prepare anessentially protein free purified carbohydrate antigen from a culture ofknown Legionella bacteria of the desired species or serogroup of aspecies.

The following examples I and II explain how the purified protein freecarbohydrate antigen is obtained.

EXAMPLE I

Bacteria of L. pneumophila Serogroup 1 (strain Philadelphia-1) wereobtained from Centers for Disease Control and Prevention (Atlanta, Ga.)and cultured on charcoal-yeast extract agar plates obtained fromNortheast Laboratory (Waterville, Me.) for a period of 72 hours at 37°C. Cells were harvested with phosphate-buffered saline (“PBS”) at pH 7.2containing 0.2% of NaN₃ and collected by centrifugation at 8000 rpm for25 minutes. The resulting cell pellet was stored at −20° C. until used.

Wet cells from this pellet were suspended in 20 ml. 0.1 M NaOH per gramof wet cells and stirred at room temperature for 45 minutes. The pH ofthe solution was then adjusted with concentrated acetic acid to 3.0 andthe solution was subjected to centrifugation at 8000 rpm for 20 minutes.The supernatant from this step was then neutralized with aqueous NaOHand dialyzed against distilled water.

The resulting dialyzate was concentrated 10 times on a rotary vacuumevaporator and then sonicated for 5 minutes in an ultrasonic bath.

Proteinase K, in a concentration of 0.2 mg. per ml. of the concentratedproduct, was added to digest the remaining proteins and the mixture wasincubated at 40° C. overnight. The next step was the addition of furtherProteinase K, in a concentration of 0.1 mg. Per ml., to the mixture,followed by further overnight incubation at 40° C. This secondincubation was followed by concentration of the product on a rotaryevaporator to a small volume, adjustment of its pH to 10-11 with 0.2%triethylamine and application of the thus-treated mixture to a column ofSephacryl S-200 from Pharmacia, equilibrated with 0.02% triethylamine.Material eluted in the first peak was pooled, adjusted with 0.1 N HCl toapproximately neutral pH and dialyzed against distilled water for 18hours followed by lyophilization.

The yield of O-polysaccharide antigen from 16.5 grams of wet cells of L.pneumophila serogroup 1 strain Philadelphia-1 was 62 mg.

It should be noted that in place of Proteinase K, any broad spectrumprotease enzyme preparation may be used. What is important in thisprocedure is the elimination of protein from the antigen to the maximumpossible extent within reasonable limits of time feasibility.

EXAMPLE II

Example I was repeated using L. pneumophila serogroup 5 strain Dallas IEas the bacteria in the culture step. 11.5 wet grams yielded 21 mg. of acarbohydrate antigen. At a later time, the procedure was again repeated,this time using L. pneumophila serogroup 5 (strain U8W) as the bacteriain the culture step.

It is within the scope of this invention to repeat the culturing andextraction steps as herein described on any other Legionella bacteriumand especially on bacteria of any other of the serogroups of L.pneumophila, i.e., on any of serogroups 2,3,4 and 6-15 inclusive, toobtain a carbohydrate antigen essentially free of proteinaceousmaterial.

In order to couple the essentially protein free O-polysaccharide productof Example 1 or the carbohydrate of Example II to a chromatographiccolumn for use in affinity purification of polyvalent antibodies to thecorresponding bacterium from which the polysaccharide or carbohydrateantigen was extracted, it is necessary to complex it with a proteinspacer molecule suitably prepared to ensure that it will stably bond toboth the polysaccharide or carbohydrate antigen and the column and willitself remain inert during the affinity purification step. A modifiedbovine serum albumin (“BSA”) was chosen as the preferred spacer moleculeand was prepared as in Example III:

EXAMPLE III Preparation of Protein Spacer Molecule

An 0.5M aqueous solution of hydrazine dihydrochloride from AldrichChemical Co., Inc. (Milwaukee, Wis.) was prepared and its pH wasadjusted to 5.2 with dry NaOH. The solution was then mixed with dry BSAfrom Sigma Chemical Co. to a final concentration of 25 mg. per ml. ofBSA. After the BSA was completely dissolved.N-(dimethylaminopropyl)-N¹-ethylcarbodiimide hydrochloride obtained fromFluka Chemical Co. (St. Louis, Mo.) was added to a final concentrationof 2.5 mg. per ml. The mixture was stirred overnight at ambienttemperature and then dialyzed for a period of about five days againstdistilled water at 4° C., with daily changes of water.

In lieu of this modified BSA, it is contemplated that other appropriatespacer molecules may be used.

The conjugation of the O-polysaccharide antigen from serogroup 1 of L.pneumophila to the spacer molecule was conducted as follows:

EXAMPLE IV Conjugation of O-polysaccharide Antigen to Spacer Molecules

L. pneumophila serogroup 1 essentially protein-free O-polysaccharideantigen was dissolved in distilled water in a concentration of 4-5 mg/mland the pH was adjusted to 5.0 with 1 M HCl. The modified BSA solutionprepared as in Example III was adjusted to pH 5.0 with 1 M HCl andslowly added, in a weight ratio of 4-to-1 of the O-polysaccharideantigen solution, to the latter. After 5 minutes of stirring, about100-200 mcl of distilled water containing theN-(dimethylamino-propyl-N¹-ethylcarbodiimide hydrochloride referred toin Example III was added in a weight ratio of 1:2 to theO-polysaccharide antigen/BSA solution. The resulting mixture was stirredat ambient temperature overnight.

To separate the conjugate of the O-polysaccharide antigen with modifiedBSA from any unreacted materials present, the reaction mixture waschromatographed on a Sepharose CL-4B column equilibrated with a bufferof 0.1 M NaH₂PO₄:0.5 M NaCl. All of the chromatography fractions weresubjected to testing for antigen activity using the commercial Binax EIAtest referred to above. All fractions that showed antigen activity inthe tested were pooled in and used for affinity column preparation asshown in Example V.

EXAMPLE V Coupling of Conjugate to Activated Chromatographic Column

An immunoadsorbent gel was prepared by conventionally activatingSepharose CL-4B with cyanogen bromide. The ligand of O-polysaccharideantigen with modified BSA, prepared as in Example IV, was coupled to itusing procedures known in the art, e.g., as described in Hermanson, G.T., et al. in Immobilized Affinity Ligand Techniques, 53-56 (AcademicPress, Inc. 1992). The gel was then packed in a column and washedsuccessively with 5-10 volumes per volume of gel with PBS of pH 7.2,triple strength PBS of pH 7.2 and 0.2 M glycine-HCl of pH 2.5. Theresulting activated column was used, as described below, for affinitypurification of polyvalent antibodies to L. pneumophila serogroup 1antigen. The column after elution should be stored in PBS or anotherneutral buffer until used.

Instead of cyanogen-bromide activated Sepharose, spherelose and variouscommercially available activated columns could be used in this step.

EXAMPLE VI Affinity Purification of Antibodies

The activated column described in Example V was used for the affinitychromatography of rabbit-anti-L. pneumophila serogroup 1 polyvalentantibodies to L. pneumophila serotype antigen according to the methoddescribed by Harlow E. and Lane, D. in Antibodies: A Laboratory Manualat 313-315 (Cold Spring Harbor Laboratory, 1988). Elution of theantibodies from the column was effected with 0.2M glycine-HCL buffer ofpH 2.5. Alternative eluants such as 3M NaSCN or 0.1M Et₃N could besubstituted.

These affinity-purified antibodies were utilized in an ICT test specificto L. pneumophila serogroup 1 as described in the following example.

EXAMPLE VII ICT Device and its Preparation

A. Preparation of Test Device

A test device comprising a hinged cardboard housing equipped with awindow to allow the viewing of both the test results and control resultswas prepared as shown in FIG. 1. The device has a recess into which isplaced a preformed plastic swab well for receiving the sample-wettedswab on the right hand (labeled 1 in the drawing). An overlabel shown inFIG. 1A is then placed over the entire right-hand side of the device.The overlabel has been equipped with two holes—a lower one (marked B onFIG. 1 a) into which the saturated swab is to be inserted and an upperone (marked A in FIG. 1A) toward which the swab will be pushed afterinsertion thereof into the hole B. The position of the overlabel withits holes A and B, and swab well cooperate to hold the swab in a properposition during the assay and to promote the expulsion of sorbed liquidfrom the swab.

A preassembled test strip (marked C on FIG. 1) described below, isinserted into the recess (labeled 2 on FIG. 1) and held in place by anadhesive applied to the bottom thereof. An overlabel shown in FIG. 1B isplaced atop the left-hand side. It has been equipped with a single hole(marked D in FIG. 1B) which mates to the right-hand side hole A when thedevice is closed for performance of the assay.

The assembled device is stored in a sealed pouch with desiccant until itis used. Prior to sealing the pouch and storing, a lightly adhesive tapeis placed on the outer edge of the right-hand half of the device.

B. Construction and Preparation of the Preassembled Test Strip

FIG. 1C shows the construction of the preassembled strip. It iscomprised of a conjugate pad of sorbent material in which a conjugate ofgold particles and the affinity-purified rabbit anti-Legionellapneumophila serogroup 1 antibodies described above have beenimpregnated. In contact with this pad is a nitrocellulose pad onto whichcapture line for the sample which reacts with the conjugate has beenestablished by embedding a stripe of affinity-purified rabbit anti-L.pneumophila serogroup 1 antibodies, prepared as described above. Thenitrocellulose pad also has a downstream control line established bystriping the pad with goat anti-rabbit immunoglobulin (IgG). Followingthe nitrocellulose pad, the strip is ended by an absorbent pad whichserves as a reservoir for liquid. All of these pads are backed by anadhesive strip when the device is ready to ship.

The conjugate pad is normally made from non-woven polyester or extrudedcellulose acetate. To prepare this pad for use in the assay, goldparticles of 50 nm. diameter are conjugated to affinity-purified rabbitanti-Legionella pneumophila serotype 1 antibodies prepared as describedabove. The conjugation is effected using a known method such as thatdescribed by DeMay in Polak, J. M. and Van Norden, S. (Eds.),Immunochemistry: Modern Methods and Application, (Wright, Bristol,England, 1986). The gold conjugate particles are mixed with a dryingagent consisting of aqueous 5 mM sodium tetraborate of pH 8.0 containing1.0% BSA, 0.1% Triton X-100, 2.0% Tween 20, 6.0% sucrose and 0.02%sodium azide. The pad is heated sufficiently to remove all of the liquidpresent and stored in a low-humidity environment pending, assembly ofthe test strip. These pads and their treatment are especially chosen sothat the pads will hold the dry conjugate and will release it only whenlater wetted by sample.

The nitrocellulose pad is first treated by embedding a stripe ofaffinity purified rabbit anti-L. pneumophila serotype 1 antibodies in afirst portion thereof, using a carrier solution of phosphate bufferedsaline. These antibodies act as the capture line. In a second portion ofthe pad downstream of the first one in the assembled test device, thecontrol line is established by striping goat anti-rabbit IgG in the samecarrier solution on the surface of the pad. The nitrocellulose pad isthen subjected to desiccation at 18-25° C. to promote permanentabsorption of the protein stripes thereto.

The absorbent pad used is of a commercially available cellulosicmaterial sold under the name Ahlstrom 939. This pad requires no specialtreatment.

C. Kit Preparation

As sold in commerce, the test device containing the finished test stripis assembled. In practice, a number of devices are packaged with acommensurate number of swabs fashioned from fibrous Dacron and a bottleof “Reagent A” equipped with a top adapted to deliver Reagent Adropwise. “Reagent A” is a solution if 2.0% Tween 20, 0.05% sodium azideand 0.5% sodium dodecyl sulfate in a 0.05M sodium citrate-sodiumphosphate buffer of pH 6.5. Positive and negative controls are alsoincluded in each kit.

The use of the finished test devices to identify L. pneumophilaserogroup 1 antigen is illustrated in the following example VIII:

EXAMPLE VIII Conducting the ICT Test for L. pneumophila Serogroup 1Antigen

In practice, the swab furnished with each device is dipped into theliquid sample, completely immersing the swab head. The use of the swabto act as a filter for undissolved solids, semisolids and colloidspresent in liquid biological samples such as urine, blood, lymph, etc.and also in liquid environmental samples is the subject of copendingapplication Ser. No. 09/044,677 of Norman Moore and Vincent Sy filedMar. 19, 1998, which is assigned to Binax, Inc. The swab is insertedinto the hole at the bottom of the device (hole B of FIG. 1A) and gentlypushed upward so that the swab tip is visible in the top hole (hole A ofFIG. 1A). The reagent A vial is held vertically above hole B and twodrops of Reagent A are slowly added. The adhesive liner is thenimmediately peeled from the right edge of the device and the device isclosed and securely sealed, thus pressing the swab in the swab wellagainst the gold conjugate pad. After 15 minutes, the result can be readin the window of the device. A negative sample—i.e., one containing noL. pneumophila serogroup 1 antigen-will exhibit only the control line inthe top half of the window. A positive sample containing the targetantigen will show two lines, the lower one of which is the patient (orsample) line; even a faint sample line indicates the presence of thetarget antigen in the sample. If no line appears in the window after 15minutes, or only a sample line appears in the lower part of the window,the test is invalid and must be repeated.

Using the procedure described above, the devices prepared as describedin Example VII were tested in the ICT procedure just described against300 patient urine samples, 100 of which had been previously diagnosed ashaving L. pneumophila serogroup 1 infection.

These ICT tests according to this invention were conducted undercircumstances such that the previous diagnoses were unknown to personnelperforming the ICT tests. Overall, 98% of the ICT tests agreed with theprevious positive diagnoses. Also overall, 98% of the urine samplespreviously diagnosed as negative for L. pneumophila serogroup 1O-polysaccharide antigen gave results in agreement therewith when testedby the ICT procedure described herein, using the ICT device described inExample VII.

EXAMPLE IX Use of the ICT to Test Environmental Samples

Applicability of this same test to environmental samples suspected ofcontaining L. pneumophila serogroup 1 was also investigated as follows:

Water was seeded with L. pneumophila serogroup 1 bacteria obtained froma commercial source. The mixture was concentrated by filtering through a0.22 μm filter. A swab dipped in the sample was applied to the device,the device was closed and the assay was allowed to proceed. A positiveresult was observed within less than 15 minutes.

EXAMPLE X Western Blot Immunoassay for Detection of Cross-ReactiveCarbohydrate Antigens of L. Pneumophila Serogroups 1, 2, 4 and 5

In order to perform the Western Blot immunoassay using a kit purchasedfrom Bio-Rad Laboratories, L. pneumophila serogroup 5 cells werecultured as in Example H. A suspension of these cells was solubilizedwith 1% sodium dodecylsulfate in the presence of 10 mM mercaptoethanolat 100° C. for 5 minutes. The solubilized cells were treated withprotease K and then subjected to electrophoretic separation of proteinaccording to standard procedures provided by Bio-Rad.

The carbohydrate antigen from L. pneumophila serogroup 5 was conjugatedto the spacer molecule described in Example III hereof in the mannerdescribed in Example IV and applied to an activated Sepharose column asdescribed in Example V. This column was then used for the affinitypurification of polyvalent rabbit antibodies specific to thecarbohydrate antigen of L. pneumophila serogroup 5 (which wereconventionally obtained from serum of a rabbit previously injected withthe protein-containing of L. pneumophila serogroup 5) using theprocedure of Example VI.

The Western immunoblot analysis was performed using a reagent kit fromBio-Rad and according to directions from this manufacturer. Briefly, thePBS extract of cells of L. pneumophila antigens 1, 2, 4 and 5 wassubjected to the SDS-PAGE in 12% polyacrylamide gel blocked with 1% BSAwith PBS transferred onto a nitrocellulose membrane. After this step,the membrane was incubated with affinity purified antibodies specific tocarbohydrate of L. pneumophila serogroup 5. The membrane, washed asrecommended by the manufacturer, and incubated with horseradishperoxidase conjugated to goat-anti-rabbit antibodies provided byBio-Rad. After washing, the membrane was developed with a substratesystem of 0.022 M 4-chloro-1 naphthol and 0.0012 M N;N-dimethyl-p-phenylene-diamine monohydrochloride in 0.1 M sodium citratebuffer of pH 6.9 containing 2.9 mM of hydrogen peroxide. FIG. 2 hereofshows the Western blot assay results compared with that of theprestained SDS-PAGE standard (in Lane 5) for the affinity purifiedantibodies of serogroup 5 of L. pneumophila against PBS extractscontaining antigens of L. pneumophila as follows:

Lanes 1 and 7—L. pneumophila serogroup 2 (strain Togus-1)

Lanes 2 and 8—L. pneumophila serogroup 4 (strain Los Angeles-1)

Lanes 3 and 6—L. pneumophila serogroup 1 (strain Philadelphia-1)

Lanes 4 and 9—L. pneumophila serogroup 5 (strain U8W).

It is pointed out that the affinity purified antibodies for Lanes 1-4were affinity purified on a column to which carbohydrate antigen from L.pneumophila serogroup 5 (strain U8W) was attached while those for Lanes6-9 were affinity purified in the same manner on a column havingattached carbohydrate antigen of L. pneumophila serogroup 5 (strainDallas IE).

FIG. 2 clearly demonstrates that affinity purified antibodies as hereindisclosed of L. pneumophila serogroup 5 react with antigens of L.pneumophila serogroups 1, 2, and 4 in addition to those of serogroup 5.

An ICT assay as described above in which affinity purified antibodiesfrom L. pneumophila serogroup 5 are substituted for affinity purifiedantibodies from L. pneumophila serogroup 1 is contemplated.

Those skilled in the art of immunochemistry generally, and especiallythose skilled in immunoassays, will recognize that other materials andingredients and at times, other procedural steps, can readily besubstituted for those specifically recommended herein. A vast array ofliterature, both patent and non-patent, discusses the design and use ofreliable, one-time-use, disposable immunoassay test devices that couldbe substituted for the preferred ICT device described and recommendedherein. It is not intended that the present invention should be limitedwith respect to substitutable assay devices, materials, ingredients orprocess steps except insofar as the following claims may so limit it.

What is claimed is:
 1. A device for determining the presence ofLegionella pneumophila serogroup 1 in a liquid sample, the devicecomprising: a porous test strip comprising a sample receiving zone;multiple binding agents, each binding agent comprising: antigen specificaffinity-purified polyclonal antibodies conjugated to a detectableparticle, wherein the antigen specific affinity-purified polyclonalantibodies specifically bind a Legionella pneumophila serogroup 1O-polysaccharide antigen and wherein the binding agents are essentiallyfree of antibodies that bind proteins of Legionella pneumophilaserogroup 1 O-polysaccharide; multiple capture agents; and a capturezone; wherein the porous test strip defines a liquid flow path extendingdownstream from the sample receiving zone and along which liquidreceived by the sample receiving zone can pass at least to the capturezone, the binding agents are disposed in a dried state along the flowpath of the test strip downstream from the sample receiving zone andupstream from the capture zone, the binding agents are mobilizable byliquid passing along the flow path, and the capture agents are disposedin the capture zone and are each capable, in the presence of Legionellapneumophila serogroup 1 in a liquid sample received by the samplereceiving zone, of binding a complex comprising one of the bindingagents bound to the antigen.
 2. The device of claim 1, wherein thecapture agents are antibodies.
 3. The device of claim 2, wherein thecapture agents are polyclonal antibodies.
 4. The device of claim 1,wherein the detectable particle is a color-producing particle.
 5. Thedevice of claim 3, wherein the detectable particle is colloidal gold. 6.The device of claim 1, wherein the detectable particle is acolor-producing particle and the capture agents are antibodies.
 7. Thedevice of claim 1, wherein the presence of Legionella pneumophilaserogroup 1 in a liquid sample is determined within fifteen minutes ofapplying the liquid sample to the device.